Mary J. Tevethia

Biology of simian virus 40 (SV40) transplantation antigen (TrAg): II. Isolation and characterization of additional temperature-sensitive mutants of SV40

Abstract Fourteen independent temperature-sensitive mutants of simian virus (SV40) were isolated following nitrous acid or hydroxylamine mutagenesis. Three mutants were assigned to the A group and seven to the BC group on the basis of standard qualitative and quantitative complementation assays. Three other mutants did not complement mutants of any complementation group well under standard conditions nor was delayed complementation observed in quantitative assays. However, these mutants were shown to complement members of the A and BC complementation groups but not members of the D group when the qualitative complementation test was modified by allowing the parental virions to uncoat at permissive temperature prior to incubation at 41°. The assignment of these mutants to the D group was substantiated by demonstrating the wild-type infectivity of DNA extracted from cells infected at 33° for growth at 41°. Thirteen of the mutants were tested for the production of tumor (T), capsid (C), virion (V), and major coat protein (VP1) antigens at permissive and nonpermissive temperature by immunofluorescence assays along with mutants which have been described previously by others for comparison. The temperature-sensitive ( ts ) mutants isolated in this study produced fully immunoreactive T antigen at both temperatures. None of the ts A mutants produced C, VPl, or V antigens at elevated temperature. The BC mutants isolated in this study all produced T antigen at 41°. These late mutants demonstrated two patterns of expression of virion antigens. One group synthesized C, V, and VP1 at 41° and were indistinguishable from wild type on the basis of antigenic phenotype. A second group showed cytoplasmic and nucleolar fluorescence for C and VPl antigens at the nonpermissive temperature similar to that observed for ts BCll previously. Mutants in this group did not produce V antigen at high temperature.

Participation of two human cytomegalovirus immediate early gene regions in transcriptional activation of adenovirus promoters.

The participation of human cytomegalovirus (HCMV) immediate early genes in the activation of the expression of adenovirus genes in trans (trans-activation) was examined. The initial strategy used was to determine the ability of HCMV genes to complement mutants of adenovirus E1a, an immediate early gene which encodes a trans-activator. The HCMV immediate early gene regions IE1 and IE2 complemented E1a-deficient mutants in three separate assays. IE1 and IE2 substituted for E1a in the synthesis of infectious adenovirus, late adenovirus RNA, and adenovirus DNA. Complementation by the IE2 gene region alone, but not by IE1 alone, was observed using the most discriminating assay, that for late adenovirus RNA synthesis. A role for both HCMV gene regions in positive transcriptional control was indicated by their ability to increase expression of chloramphenicol acetyltransferase (CAT) mediated by the adenovirus E2a promoter. The IE2 region alone activated CAT synthesis but IE1 alone had no detectable activity. Moreover, the activity of both gene regions was about 10-fold higher than that of IE2 alone. These data indicate that efficient complementation of E1a-deficient mutants and trans-activation of adenovirus early promoters involved the participation of both HCMV immediate early gene regions.

Immunoprecipitation of virus-specific immediate-early and early polypeptides from cells lytically infected with human cytomegalovirus strain AD 169

Abstract By immunoprecipitation of human cytomegalovirus-infected cell-specific polypeptides (ICPs) with a variety of human cytomegalovirus-positive sera and analysis by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, we can identify at least 20 ICSP bands from lytic infections by 6 hr postinfection (pi). Three of these polypeptide bands (78K, 73K, and 68K) may represent more than one species of polypeptide. Four polypeptide bands (78K, 77K, 73K, and 31K) can be identified as immediate-early based on their synthesis in the presence of actinomycin-D after removal from a protein synthesis block mediated by cycloheximide (CH). An immediate-early 78K polypeptide and an early 49K polypeptide are synthesized only transiently during the first 4 hr pi. Most immediate-early polypeptide synthesis is enhanced after removal of a 5 hr CH block. Taken together, these results identify many previously undetected immediate-early and early ICSPs and suggest that several regulatory events are occurring during the early phase of the lytic cycle.

Clustering of antigenic sites recognized by cytotoxic T lymphocyte clones in the amino terminal half of SV40 T antigen

The distribution of antigenic sites recognized by cytotoxic T lymphocytes (CTL) in the amino terminal half of SV40 T antigen was studied using SV40-specific CTL clones. Spleen cells of C57BL/6 (B6) mice immunized with B6/pSV3T3-20GV cells, which synthesize a truncated SV40 T antigen of amino acids 1-368, were restimulated in vitro with B6/pPVU-5-70K cells expressing SV40 T antigen of amino acids 109-708 and then cloned. The recognition sequence for all 10 CTL clones established mapped in the amino terminal half of SV40 T antigen between amino acids 109 and 271. Fine mapping of these 10 CTL clones defined three distinct antigenic sites. These three sites were abolished by the deletion of SV40 T antigen amino acids 193-211, 220-223, and 220-228, respectively. Additional CTL clones were established from spleen cells of B6 mice immunized with B6-K/S11-S24 cells, which synthesize a SV40 T antigen missing amino acids 127-250. None of these CTL clones reacted with B6/pSV3T3-20GV cells. These CTL clones recognized an antigenic site(s) which mapped in the carboxy terminal half of SV40 T antigen. Our results indicate that the antigenic sites in the amino terminal half of SV40 T antigen are tightly clustered between amino acids 193 and 271 and most probably between 193 and 228.

Biology of simian virus 40 (SV40) transplantation antigen (TrAg). IX. Analysis of TrAg in mouse cells synthesizing truncated SV40 large T antigen.

Mouse LTK- cells (H-2k) were transfected with a series of recombinant plasmids consisting of the herpes simplex virus type 1 thymidine kinase (TK) gene linked to fragments of SV40 DNA coding for portions of SV40 T antigen in pBR322, and TK+ transformants (LTK+) were selected in HAT medium. The TK+ transformants were analyzed for SV40 transplantation rejection antigen (TrAg) at the cell surface by reacting them with cytotoxic lymphocytes (CTL) generated to SV40 TrAg in C3H/HeJ (H-2k) mice. The results indicated that the cells transformed by pVBETK-1 and synthesizing full size SV40 large T antigen were efficiently lysed by SV40 CTL. In addition, cells transformed by the plasmid pVBt1TK-1 and synthesizing a truncated 33 K T antigen were also found to be susceptible to lysis by the CTL. However, LTK+ cells that were transformed with the plasmid pVBt2TK-1 and which synthesized a truncated T antigen of 12.3 K did not provide a target for SV40 CTL nor did pVBETK-1-transformed cells that did not express any of the SV40 tumor antigens. Only the pVBETK-1-transformed cells that express 94 K T antigen were able to immunize mice against a challenge of syngeneic SV40-transformed cells. These results suggest that the TrAg expression at the cell membranes of transformed cells may be associated with the proximal half of SV40 T antigen.

JC virus T'135, T'136 and T'165 proteins interact with cellular p107 and p130 in vivo and influence viral transformation potential.

The JC virus (JCV) regulatory proteins, large T antigen, small t antigen, T′135, T′136, and T′165, are encoded by five transcripts alternatively spliced from the viral early precursor mRNA. T antigen and the T′ proteins share N-terminal amino acid sequences that include the L × C × E and J domains, motifs in SV40 T antigen known to mediate binding to the retinoblastoma (Rb) proteins and Hsc70, respectively. In this study, G418-resistant cell lines were created that express wild-type or mutant JCV T antigen and T′ proteins individually or in combination. These cell lines were used to evaluate the ability of each viral protein to bind p107 and p130 in vivo, and to influence cellular growth characteristics. Differences were observed in the abilities of individual T′ proteins to bind p107 and p130 and to alter their phosphorylation status. The T′ proteins were also found to localize to the cell’s nucleus and to be phosphorylated in a cell cycle-dependent manner. JCV T antigen and T′ proteins expressed from a cytomegalovirus promoter failed to induce dense focus formation in Rat2 cells, but they did cooperate with a mutant Ras protein to overcome cellular senescence and immortalize rat embryo fibroblasts. These data indicate that, despite their sequence similarities, JCV early proteins exhibit unique activities that, in combination, effect the inactivation of cell cycle regulators, a requirement for polyomavirus-induced transformation.

Characterization of a temperature-sensitive, DNA-positive, nontransforming mutant of simian virus 40

Abstract We have characterized an early mutant of SV40, tsA1642 ( M. J. Tevethia and L. W. Ripper, 1977, Virology , 81 , 192–211 ), which differs from other tsA mutants both in its location on the genome and in its phenotype. Marker rescue experiments position tsA1642 between 0.304 and 0.325 map units in the unique coding region for large T antigen. DNA sequencing within this region reveals a single nucleotide substitution at position 1782. Unlike other tsA mutants, the tsA1642 mutation does not result in the metabolic instability of large T antigen at nonpermissive temperature, nor does it lead to a defect in autoregulation of early gene expression. At nonpermissive temperature in the lytic cycle, tsA1642 accumulates viral DNA, T antigens, and late proteins at near wild-type levels, but produces only low levels of infectious virus. In addition to its defect in lytic growth, tsA1642 is markedly defective in transformation. It is unable to transform Brown Norwegian rat kidney (B/NRK) cells to anchorage-independent growth at 40.5°. However, using the same assay, tsA1642 is nonconditionally defective in the transformation of a continuous line of C57B1/6 mouse embryo fibroblasts. TsA1642 generates transformed B/NRK cell lines some of which are temperature sensitive and some temperature resistant for the maintenance of the transformed phenotype. This mutant, therefore, appears to genetically separate, at least partially, some of the functions of large T antigen required for lytic growth from those required for transformation.

Biology of SV40 transplantation antigen (TrAg). I. Demonstration of SV40 TrAg on glutaraldehyde-fixed SV40-infected African green monkey kidney cells.

Abstract The synthesis of papovavirus SV40 transplantation antigen (TrAg) in SV40-infected permissive African green monkey kidney cells (TC7) was demonstrated by using the in vivo transplantation-rejection test. Adult BALB c mice immunized with SV40-infected cells, which had been fixed with 0.25% glutaraldehyde for 5 min at room temperature, rejected a challenge of syngeneic virus-free SV40-transformed mouse cells (VLM). Fixed uninfected TC-7 cells failed to induce transplantation immunity against VLM cells in mice. The induction of SV40 tumor immunity in mice after immunization with glutaraldehyde-fixed infected TC-7 cells was mediated by the TrAg synthesized in permissive monkey cells as a result of virus infection, rather than by the residual infectious virus in the glutaraldehyde-fixed infected cells. The treatment of infectious SV40 with 0.25% glutaraldehyde drastically reduced both viral infectivity and tumor immunity-inducing ability of the virus. Glutaraldehyde-inactivated virus also failed to induce SV40 T antigen in monkey cells, thus ruling out any possibility that the inactivated cell-associated virus was responsible for the induction of tumor immunity in vivo. The amount of infectious SV40 required to induce detectable tumor immunity was found to be 2 × 105 PFU of virus administered to adult mice, and is far greater than the amount of virus (1 × 104 PFU) which remained associated with SV40-infected monkey cells after fixation of cells with glutaraldehyde. The synthesis of SV40 TrAg in SV40-infected TC-7 cells was inhibited by 5 μg/ml of actinomycin D, but not by 15 μg/ml of cytosine arabinoside, indicating that transcription in the infected cells, but not viral-DNA synthesis, is required for the expression of TrAg. By using 0.25% glutaraldehyde to fix infected cells, it should be possible to test available temperature-sensitive mutants of SV40 for their ability to synthesize TrAg at the nonpermissive temperature, as the glutaraldehyde fixation reduces infectivity of intracellular and extracellular virus, stops the infectious cycle at a particular stage, and preserves immunogenicity of TrAg.

Complementation of an adenovirus 5 immediate early mutant by human cytomegalovirus

The initiation of the lytic cycle of adenovirus 5 requires synthesis of a transcriptional activator encoded by the viral early genetic unit, E1a. Mutant viruses lacking E1a are defective. Human cytomegalovirus activated transcription of early adenovirus genes and complemented an E1a- adenovirus mutant in cells permissive for both viruses.

Regions and Activities of Simian Virus 40 T Antigen That Cooperate with an Activated ras Oncogene in Transforming Primary Rat Embryo Fibroblasts

ABSTRACT Prolonged expression of a ras oncogene in primary cells accelerates the natural process of senescence. This ras-induced permanent growth arrest is bypassed in cells expressing the simian virus 40 large T antigen. Previously we showed that two regions of T antigen, a region consisting of the N-terminal 147 amino acids and a region consisting of amino acids 251 to 708 (T251-708), independently overcome ras-induced senescence. Coexpression of either T-antigen fragment and Ras results in the appearance of dense foci of transformed cells. Using a series of mutants that produce shorter T-antigen fragments, we show that the C-terminal limit of the N-terminal T-antigen fragment that cooperates with Ras lies between amino acids 83 and 121. The N-terminal limit of the C-terminal T-antigen fragment lies between amino acids 252 and 271. In addition, we present evidence that cooperation between the N-terminal fragment and Ras depends upon an intact T-antigen J domain and the ability of the T antigen to bind and inactivate the growth-suppressive effect of the tumor suppressor Rb. Introduction of specific amino acid substitutions surrounding residue 400 into T251-708 prevented the fragment from cooperating with Ras. T251-708 proteins with these same substitutions inhibited the transcriptional transactivating potential of p53 as effectively as did the wild-type protein. Thus, at least one activity contained within T251-708, other than inactivating p53 as a transcriptional transactivator, is likely to be required to bypass Ras-induced senescence.